Fluorine substituted alpha-azidobenzylpenicillins

ABSTRACT

A-AZIDOBENZYLPENICILLINS SUBSTITUTED IN THE PHENYL NUCLEUS WITH A FLUORINE ATOM ARE DISCLOSED. THE COMPOUNDS GIVE MUCH HIGHER AND MORE PROLONGED BLOOD LEVELS AFTER ORAL ADMINISTRATION COMPARED TO UNSUBSTITUTED A-AZIDOBENZYLPENICILLIN AND LOWER BINDING TO HUMAN SERUM COMPARED TO CHLORINE-SUBSTITUTED A-AZIDOBENZYLPENICILLINS.

United States Patent 3,555,011 FLUORINE SUBSTITUTED a-AZIDO-BENZYLPENICILLINS U.S. 'Cl. 260239.1 7 Claims ABSTRACT OF THE DISCLOSUREa-Azidobenzylpenicillins substituted in the phenyl nucleus with afluorine atom are disclosed. The compounds give much higher and moreprolonged blood levels after oral administration compared tounsubstituted u-azidobenzylpenicillin and lower binding to human serumcompared to chlorine-substituted a-azidobenzylpenicillins.

This invention relates to new antimicrobially active compounds and theirpreparation; in particular it relates to substituteda-azidobenzylpenicillins of the general formula F Na CONCHCOOH (I) andnon-toxic salts thereof, and their preparation.

The non-toxic salts of the compounds of the present invention includenon-toxic metallic salts such as sodium, potassium, calcium andaluminium salts, ammonium salts and substituted ammonium salts, e.g.salts of such nontoxic arnines as trialkylamines, includingtriethylamine, procaine, dibenzylamine, N-benzyl-fl-phenethylamine,lephenarnine, N,N dibenzylethylenediamine, dehydroabietylamine, N,Nbis-dehydroabietylethylenediamine, and other amines, which have beenused to form salts with benzylpenicillin or phenoxymethylpenicillin.

The present invention further provides a process for preparing compoundsof Formula I which process comprises reacting a compound of the formulawhere CO-X and YNH are groups of atoms capable of reacting with eachother with formation of a carbonnitrogen bond so that if necessary ordesired after hydrolysis a -CONH-group is obtained; R is a hydrogenatom, a cation or an organic radical which can be replaced by hydrogenwithout reduction of the azido group or any appreciable destruction ofthe B-lactam-thiazolidine ring system taking place.

The compound of [Formula II may be an acid chloride or its functionalequivalent as an acylating agent for a primary amino group. Suchequivalents include the corresponding acid bromides, acid azides,activated esters, acid anhydrides, including mixed anhydrides andparticularly the mixed anhydrides prepared from stronger organic acidssuch as the lower aliphatic monoesters of carbonic acid and mixedanhydrides prepared from inorganic acids such as dichlorophosphoricacid. In addition an activated heterocyclic amide such as an imidazolidemay be used or Patented Jan. 12, 1971 the free acid itself may becoupled with the 6-aminopenicillanic compound of Formula III by the useof a carbodiimide reagent or other compound such as N-ethyl-5-phenylisoxazolium-3-sulphonate, which can afford the formation of anamide upon addition to a mixture of an acid and an amine.

In the compound of Formula III, Y may be a hydrogen atom or identical toR and represents a di-(lower alkyl)- or tri-(lower alkyl)-silkyl group,the term lower meaning containing up to 6 carbon atoms.

According to one embodiment the compound of Formula III is6-aminopenicillanic acid (6-APA) or a salt thereof with an inorganicbase or an organic tertiary base and the reaction is carried out inwater in a mixture of water and an organic solvent such as dioxane,acetone, methyl isobutyl ketone, ethyl acetate, butyl acetate ordimethylformamide, in the presence of an acid-binding agent, such assodium hydroxide, sodium hydrogen carbonate or an organic tertiary base,e.g. triethylamine, N- ethylpiperidine or pyridine.

In another embodiment the compound of Formula III is 6-APA or a saltthereof with an organic base and the reaction is carried out in dryorganic solvents such as methylene chloride or chloroform in thepresence of an organic acid-binding agent such as a tertiary organicbase, e.g. triethylamine or N-ethylpiperidine.

In a third embodiment of the process of the present invention the groupR in the compound of Formula III is a di-(lower alkyl)-silyl ortri-(lower al-kyl)-sily1 radical or a tri-(lower alkyl)-tin ortriaryltin radical such as those disclosed in British patentspecification No. 1,144,191, e.g. tri- (n-butyl)-tin or triphenyltin andthe reaction is carried out in dry organic solvents, such as methylenechloride, chloroform, ethyl acetate, tetrahydrofuran, ether, benzene,toluene and dimethylformamide. After the reaction has been completed thesilyl or tin group is replaced by hydrogen by means of hydrolysisrespectively reaction with a monocarbocyclic aryl thiolate innon-aqueous solution, e.g. sodium or potassium thiophenoxide indimethylformamide, or treatment with an aqueous acid or base under mildconditions as regards pH and temperature.

According to the preferred embodiment of the process of the presentinvention the compound of Formula II is an acid chloride which isreacted with 6-APA in an aqueous organic solvent, such as aqueousacetone, dioxane or dimethylformamide or in a mixture of water and awater-immiscible organic solvent such as methyl isobutyl ketone, ethylacetate or butyl acetate. After the reaction has been completed thereaction mixture is acidified to pH 23 and extracted with an organicsolvent such as ether, ethyl acetate, butyl acetate, methyl isobutylketone or n-butanol. The penicillin is recovered from the organic phaseby extraction with Water and an inorganic base and concentrating orfreeze drying the aqueous phase or by precipitation by means of sodiumethylhexanoate and filtration.

a-Azidobenzylpenicillin (Brit. Pats. 940,488, 940,489) and substitutedanalogues of it (British Pat. 918,169) have been used as intermediatesin the preparation of the corresponding aminobenzylpenicillins. It has,however, been found that the epimer of the unsubstitutedu-azidobenzylpenicillin which has D configuration on the m-carbon in theside chain, 6-[D a azidophenylacetamido] penicillanie acid, hasexcellent antibacterial properties. (B. Sjoberg et al., AntimicrobialAgents and Chemotherapy (1967), p. 560) which makes it useful as achemotherapeutic agent.

The value of a chemotherapeutic agent is, however, not determined onlyby its antibacterial potency but also by its pharmacological propertiessuch as oral absorption, rate of excretion, and serumbinding. A goodoral absorp tion is a very desirable property as it makes it possible toobtain satisfactory blood concentrations of the chemotherapeutic agentsby the expedient oral route instead of by the more laborious anddifiicult parenteral route. Penicillins are bound to human serum proteinto varying degrees and it has been found that their antibacterialactivities diminish in proportion to their binding to the protein(Robinson and Sutherland, Brit. J. Pharmacol. (1965), 638). Kunin(Clinical Pharmacology and Therapeutics 7 (1966) 166) has found thatextensive binding of penicillins to serum markedly inhibits theirantibacterial activity in serum and that the level of unbound drug moretruly reflects the antimicrobial activity of the compound as determinedin customary in vitro assays.

Although 6-[D-u azido phenylacetamido]penicillanic acid is absorbed bythe oral route quite appreciably (L. Magni et al., Antibiotics andChemotherapy 1967, p. 5 69) we have now found that the introduction of afluorine atom into the benzene ring of the side chain surprisingly leadsto compounds which give still much better blood levels after oraladministration.

When the compounds prepared according to this invention wereadministered orally to mice all compounds were absorbed considerablybetter than 6-[D-a-azidophenylacetamido]penicillanic acid. With mandp-substituted compounds blood levels at least twice as high as thosefound with the unsubstituted compound were obtained. Longer duration ofthe blood levels was also observed.

When 6 [(-)-u-azido-m-fiuorophenylacetamido]penicilllanic acid wasadministered to humans, peak levels of the same order as those of6-[D-a-azido-phenylacetamido] penicillanic acid were found. From twohours after the administration, however, the blood levels obtained withthe fiuoro compound were considerably higher than those obtained withthe unsubstituted compound. Even eight hours after the administrationtherapeutically useful blood levels were found with the former whereasthe latter gave such ones only for six hours. When 300 mg. of the sodiumsalt of each compound were given, the blood levels of 6-[( )-uazido mfluorophenylacetamido penicillanic acid after eight hours were in factsignificantly higher than those found with 6-[D-a-azido-phenylacetamidopenicillanic acid after six hours.

These high and prolonged blood levels are of great therapeutic andpharmaceutic value as they make it possible to reduce the doses of thecompound which must be given to achieve a therapeutic effect and/or tochange the dosage regimen so that fewer intakes of the drug can be made.

The antibacterial activity of the compounds of the invention are of thesame order as that of the unsubstituted compound. As in case of thelatter the fluoro-substituted penicillins which originate fromlevorotatory side chain acids are more active than their epimersobtained from the dextrorotatory acids.

The compounds of the invention are bound to human serum to about thesame degree as 6-[D-u-azido-phenylacetamido]penicillanic acid. Some ofthe compounds are bound slightly more and some slightly less than thelatter.

Compared to the unsubstituted a-azidobenzylpenicillin the unchangedantibacterial and serum binding properties of the compounds of theinvention in conjunction with their unexpectedly much higher and moreprolonged blood levels after oral administration thus make them totherapeutically more efficient compounds.

Other in the benzene ring halogen substituted analogues of6-[D-a-azidophenylacetamide]penicillanic acid might possibly also giveenhanced blood levels after oral administration. Their use astherapeutic agents are, however, precluded by their extensive binding tohuman serum. on- Azido-m-chlorobenzylpenicillin and a azido pchlorobenzylpenicillin (e.g.) are bound to human serum to an extent of98.0% and 96.5%, leaving only 2.0% and 3.5% respectively of thepenicillins in free, active form.

4 The invention is further illustrated by the following examples.

EXAMPLE 1 Preparation of u-azido-m-fiuoro-benzylpenicillina-Azido-m-fluorophenylacetic acid (5.9 g., 0.03 mole) was dissolved intrichloroethylene (25 ml.) and treated for 5 hours at 65 C. with thionylchloride (5.5 g., 0.046 mole). The solvent was removed in vacuo and theexcess of thionyl chloride was removed by treating the residue withfresh solvent (3 X 5 ml.) which also was distilled off in vacuo.

The final residue was dissolved in dry ether (30 ml.) and added dropwiseto a stirred solution of 6-aminopenicillanic acid (6.5 g., 0.03 mole) in75% dioxane (125 ml) kept at pH 7 by addition of 2 N sodium hydroxide.When the consumption of base ceased the reaction solution was washedwith ether, acidified to pH 2 and extracted twice with ether. Thecombined ether extracts were washed with water and after addition ofwater extracted with N potassium bicarbonate (30 ml.) until the aqueousphase was neutral. Freeze drying of the latter gave the potassium saltof a-azido-m-fluorobenzylpenicillin (10 g.) with a purity of 73%(hydroxylamine assay).

a Azido m fluorophenylacetic acid, M.P. 63-64" C. (Found (percent): C,49.40; H, 3.26; F, 9.85; N, 21.26. Calcd. for C H FN O (percent): C,49.23; H, 3.10; F, 9.74; N, 21.53) was obtained froma-bromo-m-fluorophenylacetic acid, M.P. 65 C. (Found (percent): C,41.07; H, 2.56; Br, 34.22; F, 8.33. Calcd. for C H BrFO (percent): C,41.23; H, 2.60; Br, 34.29; F, 8.15) by reaction with sodium azide inacetone in presence of sodium carbonate.

This product was found to inhibit the growth of Staph. aureus, Oxford ata concentration of 0.01 meg/ml.

EXAMPLE 2 Preparation of 6-[( )-a-acido-m-fluorophenylacetamido]penicillanic acid (a) The laevorotatory enantiomer ofa-azido-m-fluorophenylacetic acid (2.6 g., 0.0136 mole) intrichloroethylene 15 ml.) was treated for 5 hours at 65 C. with thionylchloride (2.5 g., 0.021 mole). The solvent was evaporated in vacuo andthe excess of thionyl chloride was removed by treating the residue withfresh solvent (3X 5 ml.) which also was distilled off in vacuo. Thefinal residue was dissolved in dry ether (30 ml.) an added dropwise to astirred and ice-cooled mixture of 6-aminopenicillanic acid (2.94 g.,0.0136 mole) in 75% dioxane ml.) kept at pH 7 by addition of 2 N sodiumhydroxide.

After the consumption of base had ceased the mixture was diluted withwater, washed with ether, acidified to pH 2 and extracted twice withether. The combined ether extracts were washed with water and extractedwith N potassium bicarbonate solution (13 ml.) until the aqueous phasewas neutral. Freeze-drying of the latter gave the potassium salt of 6-[)-a-azido m fluorophenylacetamido]penicillanic acid (3.0 g.) with apurity of 63% (hydroxylamine assay). Precipitation from methanol-ethergave a product which analyzed 65% and had an [a +157 (c.=0.1, H O).

This product was found to inhibit the growth of Staph. aureus, Oxford ata concentration of 0.03 meg/ml.

a Azido m fluorophenylacetic acid [u -101.1 (c.=0.9, ethanol) wasobtained by fractional crystallization of the pseudoephedrine salt ofracemic aazido-m-fiuorophenylacetic acid from methanol/ether.

(b) To 6-aminopenicillanic acid (95.6%, 200 g., 0.885 mole) dissolved inwater (15 l.) and adjusted to pH 7-7.5 by addition of 5 N sodiumhydroxide 4-methyl-pentanone- 2 (7.5 1.) was added and pH was adjustedto 3 by addition of 5 M sulphuric acid.

A solution of ()-u-azido-m-fluorophenylacetyl chloride intrichloroethylene (1.07 mol/kg.; 914 g., 0.977

mole) was added at 8-11 C. during 15 minutes while the mixture wasvigorously stirred and kept at pH 3 by addition of 5 N sodium hydroxide.After stirring for further 20 minutes filtered (Celite) was added andthe mixture was filtered. The organic phase was recovered and afterdrying over anhydrous sodium sulphate treated with 2 N butanolic sodium2-ethylcaproate (532 ml.). After stirring for 60 minutes the precipitateformed was collected by filtration, washed thoroughly with4-methylpetanone-2 and dried in vacuo yielding the sodium salt of '6-[)-a-azido-m-fluorophenylacetamido] penicillanic acid (245 g.) with apurity of 93.1% (alkalimetric assay).

A part of the product (10 g.) was recrystallized from isopropanol-waterto give 8.3 g. of the compound with a purity of 94.1% and a [a =+189,(c.=0.25, H O).

EXAMPLE 3 Preparation of 6-[)-u-azido-m-fluoro-phenylacetamido]penicillanic acid Starting with thedextrorotatory enantiomer of a-azidom-fluorophenylacetic acid (2.6 g.,0.0136 mole) and repeating the procedure of the preceeding examples thepotassium salt of 6-[(+)-a-azido-m-fluorophenylacetamido] pencillanicacid (4.0 g.) with a purity of 58.3% (hydroxylamine assay) was obtained.Precipitation from methanolether gave a product with a purity of 65% and+244 (c.=0.1, H O).

This product was found to inhibit the growth of Staph. aureus, Oxford ata concentration of 0.06 mcg./ml.

a Azido m fluorophenylacetic acid [0: +100.4 (c.=0.25, ethanol) wasobtained by fractional crystallization of the l-ephedrine salt ofracemic a-azidom-fluorophenylacetic acid from ethyl acetate/diethylether/petroleum ether.

EXAMPLE 4 Preparation of a-azido-o-fluoro-benzylpenicillina-Azido-o-fluorophenylacetyl chloride (3.6 g., 0.017 mole) in drydioxane (5 ml.) was added dropwise to a stirred and ice-cooled mixtureof -6-aminopenicillanic acid (4.5 g., 0.02 mole) in 75% dioxane (50ml.), the pH being maintained at 6.5 by automatic addition of 2 N sodiumhydroxide. When consumption of base ceased the reaction solution waswashed with ether, acidified to pH 2 and extracted three times withether. The combined ether extracts were washed with water and afteraddition of water extracted with N potassium bicarbonate until theaqueous phase was neutral. Freeze-drying of the latter gave thepotassium salt of a-azido-o-fluorobenzylpenicillin (5.0 g.) with apurity of 64% (hydroxylamine assay).

This product was found to inhibit the growth of Staph.

aureus, Oxford at a concentration of 0.03 mcg./ml.

cc Azido o fluorophenylacetic acid, M.P. 534 C. (Found (percent): C,48.88; H, 3.60; F, 9.68; N, 21.38; equiv. weight 197. Calcd. for C H FNO (percent): C, 49.23; H, 3.10; F, 9.74; N, 21.53; equiv. weight, 195)was obtained from the corresponding a-bromo acid by reaction with sodiumazide in 95% acetone in presence of sodium carbonate. It was convertedinto its acid chloride by reaction with thionyl chloride for 45 min. at80 C.

EXAMPLE 5 Preparation of u-azido-p-fiuorobenzylpenicillin (a) u-AZidO pfluorophenylacetyl chloride (13.3 g., 0.062 mole) was added dropwise toa stirred and icechilled mixture of 6-aminopenicillanic acid (16.2 g.,0.075 mole) in 75% dioxane (350 ml.) maintained at pH 6.5 by automaticaddition of 2 N sodium hydroxide. When consumption of base ceased thereaction solution was washed with ether, acidified to pH 2 and extractedtwice with ether. The combined ether extracts were washed latter gavethe sodium salt of a-azido-p-fiuorobenzylpenicillin (21.5 g.) with apurity of 78%.

This product was found to inhibit the growth of Staph. aureus, Oxford ata concentration of 0.06 mcg./ml.

oz Azido p fluorophenylacetic acid, M.P. 634 C. (Found (percent): C,49.14; H, 3.52; F, 9.61; N, 21.30. Calcd. for C H FN O (percent): C,49.24; H, 3.10; F, 9.74; N, 21.53) was prepared from the correspondingabromo acid by treatment with sodium azide in 95 acetone in presence ofsodium bicarbonate. The acid chloride, B.P. l2l122 C./ 8 mm., wasobtained by treatment of the acid with thionyl chloride.

(b) 6-aminopenicillanic acid 1.1 g., 0.005 mole) in dry methylenechloride (50 ml.) was treated with triethylamine (1 g., 0.01 mole) andstirred for 1 hour in the ice bath. p-Nitrophenylu-azido-p-fiuorophenylacetate (1.25 g., 0.004 mole), dissolved in drymethylene chloride (10 ml.) was added and stirring was continued for 20hours without cooling. The reaction mixture was washed twice with dilutesulphuric acid at pH 2 and then with water. The organic layer was thenextracted with dilute sodium hydroxide until the aqueous phase reachedpH 6.3. The aqueous solution was separated and washed with ether andthen evaporated in vacuo at 30 C. to give the sodium salt ofa-azido-p-fluoro-benzyl penicillin (1.25 g.) with a purity of 66%(hydroxylamine assay).

The p-nitrophenyl a-azido-p-fluorophenylacetate was obtained bytreatment of the a-azido-p-fluorophenylacetic acid in methylene chloridewith p-nitrophenol in presence of dicyclohexyl carbodiimide.

(c) a-A2.ido-p-fluorophenylacetic acid (1 g., 0.005 mole) in drytetrahydrofurane (30 ml.) was stirred and treated withN,N-carbonyl-diimidazole (0.9 g., 0.0055 mole) and kept for 1 hour at 50C. After cooling to room-temperature a dimethylsilyl derivative of6-aminopenicillanic acid in ethylacetate, obtained as described below,'was added and stirring was continued for 2 hours. Water (50 ml.) wasadded and pH was adjusted to 7. The mixture was washed with ether andthe aqueous phase was acidified to pH 2 and extracted twice with ether.The combined ether extracts were washed with water and then extractedwith N potassium bicarbonate until the aqueous phase was neutral.Evaporation of the latter gave the potassium salt ofa-azido-p-fluorobenzyl penicillin (1.8 g.) with a purity of 63%(hydroxylamine assay).

The dimethylsilyl derivative of 6-amino-penicillanic acid was preparedas follows: To 6-amino-penicil lanic acid (1.1 g., 0.005 mole) suspendedin dry ethyl acetate (30 ml.) triethylamine (1.5 g., 0.015 mole) wasadded with stirring at 80 C. followed by dimethyl-dichlorosilane (0.65g., 0.005 mole), dissolved in dry ethyl acetate (5 ml.). After 5 min.the precipitate formed was removed by filtration under dry conditionsand the filtrate was cooled to room-temperature and used directly forthe synthesis described above.

(d) a-Azido-p-fluorophenylacetic acid (1 g., 0.005 mole) andtriethylamine (0.5 g., 0.005 mole) in dry dimethylformamide (10 ml.)were stirred at 10 C. and treated dropwise with ethyl chloroformate(0.54 g., 0.005 mole) dissolved in dry ether (5 ml.) 10 min. after theaddition was completed a solution of 6-aminopenicillanic acid (2.1 g.,0.01 mole) and triethylamine (1.1 g., 0.011 mole) in water (10 ml.) wasadded and stirring was continued for 2 hours without external cooling.The reaction mixture was washed with ether, acidified to pH 2 andextracted three times with ether. The combined ether extracts Werewashed with water and extracted with N potassium bicarbonate until theaqueous phase was neutral. Evaporation of the latter in vacuo atroom-temperature gave the potassium salt of a-azido-p-fluoro-benzylpenicillin (1.7 g.) with a purity of 53% (hydroxylamine assay).

(e) A stirred and ice-cooled solution of a-azido-p-fluorophenylaceticacid (1 g., 0.005 mole) in dry methylene dichloride (15 ml.) was treatedwith an ice-cooled solution of tri-n-butyl -6-aminopenicillanate (2.5g., 0.005

mole) in dry methylene dichloride (15 ml.) immediately followed by alikewise ice-cooled solution of dicyclohexyl carbodiimide (1.03 g.,0.005 mole) in dry methylene dichloride (7 ml.). Stirring was continuedover night at +4 C. Ethyl acetate (50 ml.) was added and the mixture wasfiltered. The filtrate was stirred with water (55 ml.) and adjusted topH 6.5 for 10 min., filtered, dried over anhydrous magnesium sulphateand evaporated to dryness.

The residue was reprecipitated from benzene-petrolether to give 2.1 g.of product, which was dissolved in dry dimethylformamide (2.5 ml.) andtreated for 30 min. with potassium thiophenolate (0.46 g.). The mixturewas poured into dry ether while stirring giving a precipitate of thepotassium salt of a-azido-p-fluorobenzyl penicillin (1.2 g.) with apurity of 79% (hydroxylamine assay).

(f) a-Azidop-fluorophenylacetic acid (1 g., 0.005 mole) in drytetrahydrofuran (25 ml.) was stirred and cooled to 20 to 30 C. andtreated dropwise with phosphorus oxychloride (0.75 g., 0.005 mole) andthen with triethylamine (0.05 g., 0.005 mole) each dissolved in a fewml. of dry tetrahydrofuran. A solution of a trimethylsilyl derivative of6-APA in tetrahydrofuran (20 ml.) prepared as described below andtriethylamine (0.5 g., 0.005 mole) was added and stirring was continuedfor 2 hrs. first at 10 to 20 C. and at the end of the reaction at C.After addition of water and adjustment of the pH to 7 the mixture waswashed with ether, acidified to pH 2 and extracted twice with ether. Thecombined ether extracts were washed with water and extracted with a Npotassium bicarbonate solution until the aqueous phase was neutral.Evaporation of the latter in vacuo at room-temperature gave thepotassium salt of a-azido-pfiuoro-benzyl penicillin (1.7 g.) with apurity of 39% (hydroxylamine assay).

The trimethylsilyl derivative of 6-amino penicillanic acid was preparedas follows: 6-aminopenicillanic acid (1.1 g., 0.005 mole) was treatedwith N-trimethylsilyldiethylamine (3.6 g., 0.025 mole) at 80 C., thediethylamine formed being continuously removed by distillation. After 30min. the excess of the silylating agent was removed in vacuo and theresidue was dissolved in dry tetrahydrofuran (20 ml.) and used for theabove reaction.

EXAMPLE 6 Preparation of 6-[ a-azidop-fluorophenylacetamido]penicillanic acid The laevorotatory enantiomer ofa-azido-p-fiuorophenylacetic acid (4.7 g., 0.024 mole) intrichloroethylene (20 ml.) was treated 2.5 hrs. at 65C. with thionylchloride (4.4 g., 0.037 mole). Solvent and excess thionyl chloride wereevaporated in vacuo and the residue was dissolved in dry ether (20 ml.)and added dropwise to a stirred and ice-chilled mixture of6-aminopenicillanic acid (7.8 g., 0.036 mole) and 75% dioxane (75 ml.)maintained at pH 7.0 by addition of 2 N sodium hydroxide. After theconsumption of base had ceased the mixture was diluted with Water,washed with ether, acidi- 8 phase was neutral. Freeze-drying of thelatter gave the potassium salt of6-[(-)a-azido-pfiuorophenylacetamido]penicillanic acid (6.5 g.) with apurity of 78% (hydroxylamine assay).

This product Was found to inhibit the growth of Staph. aureus, Oxford ata concentration of 0.01 meg/ml. Recrystallization from isopropanol/ethergave a product with a purity of 86% and [a] =+163 (c.=0.25, H O),

( a-AZido-p-fiuorophenylacetic acid,

[a l1l.0 (c.=0.25, ethanol) was obtained by fractional crystallizationof the ephedrine salt of racemic a-azido-p-fluorophenylacetic acid.

EXAMPLE 7 Preparation of 6- -a-azido-p-fluorophenylacetamido]penicillanic acid In the manner described in Example 6, starting withthe dextrorotatory enantiomer of u-azido-p-fluorophenylacetic acid (3.9g., 0.02 mole) the potassium salt of 6-a-azido-pfluorophenylacetamido]penicillanic acid (5.5 g.) with a purityof (hydroxylamiue assay) and [a =+279 (c.=0.25, H O) was obtained.

This product was found to inhibit the growth of Staph. aureus, Oxford ata concentration of 0.03 mcg./ ml.

(+ )-a-Azido-p-fluorophenylacetic acid,

[oc +110 (c.=0.25, ethanol), was obtained by fractional crystalizationof the d-amphetamine salt of the racemic u-azido-pfiuorophenylaceticacid.

PHARMACOLOGICAL TESTS The products prepared according to the precedingexamples, 6-[D a azido-phenylacetamido]penicillanic acid,a-azido-m-chlorobenzylpenicillin and a-azido-pchlorobenzylpenicillinwere tested giving the results found below. The compounds are designatedas follows: I denotes 6-[ )-a-azido-m-fluorophenylacetamido]-penicillanic acid II denotes 6-[ +)a-azido-mfluorophenylacetamido]-penicillanic acid III denotes a-azido-m-fiuorobenzylpenicillin, i.e. amixture of I and II obtained according to Example 1 from DL-u-azidom-fluorophenylacetic acid IV denotes 6-[-a-azido-p-fluorophenylacetamido] penicillanic acid V denotes 6-[(+)-aazido-pfiuorophenylacetamido]- penicillanic acid VI denotesa-azido-p-fluorobenzylpenicillin, i.e. a mixture of IV and V obtainedfrom DL-a-azido-p-fluorophenylacetic acid VII denotesa-azido-o-fiuorobenzylpenicillin VIII denotes6-[D-a-azido-phenylacetamido]penicillanic acid IX denotesa-azido-m-chlorobenzylpenicillin X denotesu-azido-pchlorobenzylpenicillin (a) Antibacterial activity (MinimumInhibitory Concentration M.I.C. mcg./ml.).

Compound I II III IV V VI VII VIII IX X Purity organism. 65 100 78 69 8473 99 81 Dipl, gmeum 0.006 go. 006 0. 006 0. 01 S0. 006 go. 006 S0. 006S0. 032 .Nezss catarrh go. 006 0. 03 0. 01 0.13 0. 01 0. 01 o. 01 Staph.aureus, 0mm. 0. 01 0. 06 0. 01 0. 01 0. 03 0. 0s 0. 03 go 005 0. 01 6.01 Strept. face. 2. 5 0. 63 0. 63 2. 5 l. 25 1. 25 0. 63 0. 25 0. 63Sircpt. Py o go. 006 S0 006 go. 006 go 006 go. 006 go. 006 go 006 S0.006 S0. 006 Slrcpt vlrzdans go. 006 S0. 006 S0. 006 go. 006 $0. 006 $0.006 S0. 006 0.06 0. 03 25(8))6D5e6tgef or binding to hyman serumdetermined by an ultrafiltration technique (Robinson and Sutherland,Brit. J. Pharmaeo fied to pH 2 and extracted with ether four times. Thecombined ether extracts were washed with water and extracted with Npotassiurn'bicar'bonate until the aqueous (b) Degree of binding to humanserum determined by an ultrafiltration technique (Robinson andSutherland, Brit. J. Pharmacol. 25 (1965) 638).

(c) Blood concentration (mcg. ml.) in mice after oral application of 200mg. penicillin/kg. bodyweight.

Compound IV V VII I II VIII (d) Blood concentrations in humans.

Capsules containing sodium6-[(--)-u-azido-m-fluorophenylacetamido]penicillanate (I, 300 mg.) weregiven to six healthy, fasting volunteers. Blood samples were drawn after0.5, 1, 2, 4, 6 and 8 hours and analyzed for penicillin content. Threedays later sodium d-[D-u-azido-phenylacetamido1penicillanate (VIII, 300mg.) were given to' the same subjects under the same conditions. Thefollowing mean blood concentrations (mcg./ ml.) with standard errors ofmean were found.

Time, hr.

Compound:

I 9. 16 7. 45 3. 24 0. 99 0. 34 0. 125 :22. 74 :IzO. 68 :l:0. l3 :EO.075 5:0. 015 :110. 008

VIII 9. 55 7. 64 2. 38 0. 36 0. 081 0. 027 =l=1. 99 i=0. 85 :bO. 5 5:0.0032 =|=0. 0098 The differences between the mean values of the twocompounds were not significant at 0.5 and 1.0 hour, but the higher bloodlevels observed with the fluoro compound were significant (p0.01) after2.0 and 4.0 hours and highly significant (p 0.00 1) after 6.0 and 8.0hours. The 8 hr.-value for I was significantly higher than the 6 hr.-value for VIII (p 0.05).

We claim:

1. A compound selected from the group consisting of and non-toxic saltsthereof.

2. The compound according to claim 1 which is in the form of anoptically pure isomer.

3. A compound selected from the group consisting of racemic a-azido rnfluorobenzyl-penicillin and its nontoxic salts.

4. A compound selected from the group consisting of racemicu-azido-rn-fiuorobenzyl-penicillin and its non-toxic salts.

5. A compound selected from the group consisting of racemica-azido-p-fluorobenzyl-penicillin and its non-toxic salts.

6. A compound selected from the group consisting of 6-[()-a-azido mfluorophenylacetamido]penicillanic acid and its non-toxic salts.

7. A compound selected from the group consisting of 6-[()-a-azido pfluorophenylacetamido]penicillanic acid and its non-toxic salts.

References Cited UNITED STATES PATENTS 3,293,242 12/1966 Sjoberg et a1260239.1 3,362,952 1/ 1968 Stove et al. 260239.1

NICHOLAS S. RIZZO, Primary Examiner US. Cl. X.R. 424-271 3 93 UNITEDstuns PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 555,011Dated Januarx l2 1971 Inventor(s) Bertil Ake Ekstrom and Berndt OlofHarald Sjoberg It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 1, line 53, "YNHCHC" should be -YNHCHCH-;

Column 2, line 9, "silkyl" should be -silyl;

Column 2, line 14, after "water" insert or--;

Column 3, lines 29-30 "penicilllanic" should be penicilla1 Column 3,lines 40 & 42 after "fluorophenylacetamide" inser- Column 5, line 4"filtered" should be filteraid-;

Column 5, line 10, "petanono" should be pentanone;

Column 7, line 20, (0.05 g. should be (0.5 g.

Column 7, line 68, "hyman" should be -human;

Column 7, line 68, "Pharmaco" should be Pharmacol;

Column 8, line 8, [011 should be [01 7 Column 10 line 20, claim 3, "m"should be o- Signed and sealed this 26th day of October 1971.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer ActingCommissioner of. Pat

